The goal of this project is to elucidate the mechanisms by which interleukin-32 (IL-32) enhances immunity against Mycobacterium tuberculosis (MTB). A comprehensive understanding of IL-32 action will not only advance our knowledge of the host-protective immune response to tuberculosis (TB), but also drive future investigations designed to enrich vaccine therapy and cultivate innovative, immune-modifying treatments. IL-32 is a recently described cytokine. We have shown that IL-32 is induced by MTB infection and that it protects host cells against MTB. We discovered that IL-32 inhibits intracellular growth of MTB in THP-1 macrophages through caspase-3-dependent apoptosis and yet-to-be identified caspase-3-independent cell death pathways. While IL-32 plays a potentially critical role in controlling TB, important questions remain. We hypothesize that IL-32 reduces viability of intracellular MTB by inducing macrophages to undergo various types of programmed cell death (PCD), including caspase-3-dependent and caspase-independent apoptosis as well as pyroptosis, a unique form of inflammatory PCD mediated by caspase-1 and inflammasome complexes. Both apoptosis and pyroptosis are known killing mechanisms of intracellular pathogens including mycobacteria. While a mouse homolog of IL-32 has not been found, murine macrophages are activated by human IL-32. RAW 264.7 mouse macrophages incubated with IL-32 have a lower burden of intracellular MTB. To study the anti-TB effect of IL-32 in vivo, we generated transgenic mice that express IL-323 in the lungs under the control of the SPC promoter (SPC-IL-323Tg). Aerosol infection with the hypervirulent MTB W-Beijing HN878 showed that the SPC-IL-323Tg mice have reduced bacterial burden in the lungs and spleen, a more protective immune phenotype, and increased survival compared to wild type (WT) C57BL/6 mice. Recently, our collaborators developed a Tg mouse strain in which IL-323 is under the control of the 2-actin gene promoter. IL-32 is widely expressed in the tissues of these mice, including the lung, spleen, and white blood cells. To begin to address the clinical relevance of IL-32 in humans, we will test the efficacy of IL-32 against a virulent clinical strain of MTB in primary human alveolar macrophages (AM). We propose to more fully describe the mechanisms by which IL-32 kills intracellular MTB as well as the complex immunological pathways affected by IL-32 through the following three aims. Aim 1. Elucidate how programmed cell death pathways contribute to IL-32 induced inhibition of intracellular MTB. We hypothesize that caspase-3-independent apoptosis and caspase-1-mediated pyroptosis are additional mechanisms by which IL-32 exerts its anti-TB effects. Aim 2. Determine how IL-32 transgenic mice are protected against MTB infection. We hypothesize that SPC-IL-323Tg mice will have sustained resistance to MTB at longer times of infection, 2-actin-IL-323Tg mice will also resist MTB infection, and both knock-in models will survive longer and exhibit a more protective immune response with greater induction of PCD in the AM than infected WT mice. Aim 3. Define the anti-mycobacterial activity of IL-32 in primary human cells and determine whether the anti-TB effects of IL-32 can be enhanced. We hypothesize that IL-32 will antagonize intracellular MTB in primary human AM due to its ability to induce caspase-3-dependent apoptosis and caspase-1-mediated pyroptosis. Since W-Beijing strains are less likely to induce host cell apoptosis, we hypothesize that IL-32 will be more efficient in killing MTB H37Rv than W-Beijing HN878. Additionally, enhancing the bioactivity of IL-32 should increase induction of PCD and MTB killing.